In the last decades a plurality of analytical methods using chromatographic technology has been provided. Further refined methods have been developed by coupling two chromatographic techniques. An example is comprehensive two-dimensional gas chromatography (GC×GC), in which usually a long nonpolar GC column is coupled with a short polar GC column, with the aim to improve the so-called peak capacity, which is a performance measure describing the number of resolved signals of the analytical separation in a specified time slot. The separation on the long column leads to typical retention times in the minute range, while fast separations with orthogonal selectivity can be realized in the second range on the short column. The results of the GC×GC technique can be presented as so-called contour plots, wherein signal intensities are assigned to the retention times of the first and second separation dimension on the basis of a color scale.
In analogy to the GC×GC, other two-dimensional chromatography systems have been described for separations with liquid mobile phases. Two-dimensional liquid chromatography (LC×LC) has found increased use [1]. Recently, a combination of liquid chromatography (LC) and chip electrophoresis has been described [2]. Also known are two-dimensional separations of various electrophoretic separation methods as well as the coupling of ion chromatography (IC) and reversed phase liquid chromatography [3].
For the separation of ionic species ion chromatography (IC) and capillary electrophoresis (CE) are the major instrumental techniques. Both separation methods are based on completely different separation mechanisms.
Ion chromatography is a method that allows the separation of ions and protonated/deprotonated polar molecules based on their affinity to an ion exchanger.
Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge and the hydrodynamic radius of an ionic species. It has been shown that CE separations can be carried out in short fused silica capillaries in conjunction with mass spectrometry in the migration time range of a few seconds [4].
Although two-dimensional systems were known, a combination of ion exchange chromatography methods and electrophoresis based methods was not contemplated in the past for various reasons. In particular, the relatively slow separation speed of conventional CE based on the use of long capillaries was deemed to exclude the construction of a corresponding two-dimensional separation system. A difference between both systems is the fundamentally different flow characteristic that develops in the injection cell around the CE separation capillary.
One crucial aspect for the technical realization of two-dimensional separations with the conversion of all sample components from the first to the second dimension of separation is the use of a modulator which controls the transfer from the first to the second dimension. In DE 19717738C1 [5] it has been found that with the process of capillary batch injection analysis (CBIA) small sample volumes in the nanoliter range, which are handled by means of a capillary coupled to a microliter syringe, may be injected directly onto the surface of a sensor in a detection cell filled with electrolyte solution, and the small injected sample volumes may then be dispersed in the electrolyte reservoir by a stirrer, such that the measurements can be repeated at a high frequency, showing only a negligible base line drift due to the large dilution in the electrolyte reservoir. It has been shown later that the CBIA concept can also be adapted as an injection concept for CE [6]. However, both in the classical CBIA as well as in the case of CBIA-CE, only discrete sample volumes can be taken up by means of a capillary and then injected to a sensor surface or to the inlet of a CE capillary, respectively.
Batch processing is labor-intensive and time consuming, as for every batch, the system has to be cleaned and the solutions have to be prepared before the system is ready for the next batch. Furthermore, batch processing is more error-prone due to being labor-intensive, and comparison of the results of the analysis of the different batch samples can vary due to the practically separate experimental setup conditions for every batch.
It was an objective of the present invention to provide a device and a method for an improved separation of ionic species, which has increased peak capacity, is more cost-effective, and less error-prone than the methods known and used in conventional manner.